A Protein A column is commonly used in many of the purification processes used for the production of antibody drugs. The use of Protein A column has the advantage of having production with high purity at the early stage of the process. However, the cost of Protein A is 30 times more expensive than that of other commonly-used ion exchange resins, resulting in a high production costs for antibody drugs.
According to previous reports, the Protein A resin accounts for about 35% of the raw material costs associated with antibody drug production (Journal of Chromatography A, 1024 (2004) 79-85), and trace amounts of Protein A remaining in the antibody sample may cause immunogenic or other physiological responses when administered to humans (Purification Tools for Monoclonal Antibodies, Validated Biosystems, Tucson, Ariz., 1996). Therefore, purification processes using Protein A column require constant monitoring and removal of residual Protein A in each purification step. In addition, since Protein A works based on its bioaffinity towards the target, it has the disadvantage of low chemical stability. Thus, to maintain the bioactivity of Protein A, 1 M NaOH cannot be used even though it is essential for cleaning the column to make it available for subsequent use. Without using 1 M NaOH, it is hard to completely remove impurities from the column, and thus the number of times that the column can be re-used is substantially lower than the number allowed by the use of chemical resins.
Despite the high cost, major global pharmaceutical companies prefer using Protein A for purification of antibodies because it can achieve high purity of antibodies at the early stage. For instance, Genentech produces an antibody drug, namely Herceptin which is purified through a process utilizing Protein A, a cation exchange resin, an anion exchange resin, and a hydrophobic interaction resin (HIC) (Sven Sommerfeld a, Jochen Strube b, Chemical Engineering and Processing 44 (2005) 1123-1137). However, due to the disadvantages of Protein A described above, a more efficient and cost-effective purification method for antibodies still needs to be developed.
In order to develop an antibody purification method that does not use Protein A column, the first requirement for any alternative method is that it removes impurities to an equal or greater than Protein A. Especially when a CHO cell line is used as a host cell for producing the antibody, the cell culture contains not only the desired antibodies, but also a large amount of impurities including host cell proteins (HCPs), host cell-derived DNAs (HCDs), and growth factors. Therefore, in any case not using Protein A for antibody purification, successful removal of impurities at the early stage is an important factor due to the high amount of impurities.
To solve this problem, Warren Schwartza et al. have developed an antibody purification method that uses MEP HyperCel which is a type of hydrophobic charge induction chromatography (HCIC) (Journal of Chromatography A, 908 (2001) 251-263). In this method, antibodies having high purity are isolated without ultrafiltration from the cell culture containing salts. However, HCIC is still 2 to 5 times more expensive than the use of ion exchange resins. Also it has not been found whether the removal of impurities through HCIC is more effective than using ion exchange resins. Meanwhile, Egisto Boschetti has proposed using a thiophilic chromatography (T-gel) that is based on the chemical affinity of resin for producing an antibody having high purity, whereas Protein A chromatography is based on its own bioaffinity (J. Biochem. Biophys. Methods 49 (2001) 361-389). However, T-gel is still not as effective as Protein A for preparing antibodies having high purity and also its cost is 5 times higher than that of the method using ion exchange resins. Therefore it has not been used in the industry as of yet.
Meanwhile, as another purification method, an ion exchange resin such as a cation exchange resin can be used in an adsorption column. Examples of cation exchange resins that are currently used in the industry include CM Fast Flow and SP Fast flow manufactured from GE-healthcare, and Fractogel SO3 and Fractogel COO− manufactured from Merck (US2007029244). However, a cation exchange resin still has the limitation of inefficient removal of impurities at the early stage. Likewise, optimizing the numerous conditions of the purification process is the key for a successful development of a novel purification process.
In the antibody purification process, maintaining the consistency of quality is as important as the removal of impurities. An antibody consists of two heavy chains and two light chains which are linked by disulfide bonds, while the Fc portion of the heavy chain is glycosylated. However, the antibodies produced in CHO cells used as a host cell include various isoforms (Hongcheng Liu, Georgeen Gaza-Bulseco, Journal of Chromatography B, 837 (2006) 35-43). Most of the isoforms have few amino acid modification such as deamidation, oxidation, and the like (Isamu Terashima, Akiko Koga, Analytical Biochemistry 368 (2007) 49-60). Especially, if the complementarity determining region (CDR) being antigen-binding site, is modified by deamidation thereby forming antibody isoforms, there is a reduction in the binding affinity of antigen to antibody, thereby affecting the bioactivity of the antibody (Reed J. Harrisa, Bruce Kabakoff, Journal of Chromatography B, 752 (2001) 233-245).
Various types of antibody isoforms can be formed, for example, by deamidation of asparagine yielding aspartate (Boxu Yan, Sean Steen, Journal of Pharmaceutical Sciences, Vol. 98, No. 10, October 2009), and by oxidation of methionine yielding methionine sulfate (Chris Chumsae, Georgeen Gaza-Bulseco, Journal of Chromatography B, 850 (2007) 285-294). In addition, glutamate at the N-terminal of heavy chains may be transformed into pyroglutamate by forming a five-member ring structure (William E. Werner, Sylvia Wu, Analytical Biochemistry 342 (2005) 120-125). Since these isoforms affect the bioactivity of antibodies, the proportion of isoforms present in the antibody needs to be controlled during the antibody production process.
For regulating the amount of antibody isoforms, a cation exchange chromatography can be used since it can isolate the desired antibodies adsorbed to the column by desorbing them based on the difference in net charge but an appropriate purification condition needs to be specified as well.